Severity of disease was classified according to the intensity of the immunosuppressive regimens applied and compared to gene copy numbers, too
Severity of disease was classified according to the intensity of the immunosuppressive regimens applied and compared to gene copy numbers, too. of disease was classified according to the intensity of the immunosuppressive regimens applied and compared to gene copy numbers, too. In addition, we performed a TaqMan based analysis of three lupus-associated single-nucleotide polymorphisms (SNPs) located inside the major histocompatibility complex (MHC) to investigate the independence of complement in association with SLE. Results Homozygous deficiency of the isotype was identified as the strongest risk factor for SLE (odds ratio (OR)?=?5.329; were associated with SLE (OR?=?3.699; gene copy numbers in patients, the mean concentration ranging from 0.110?g/l (two copies) to 0.256?g/l (five to six copies; and homozygous deletion of were associated with a disease course requiring cyclophosphamide therapy (OR?=?4.044; gene with its isotypes and and the special feature of copy number variation has been repeatedly studied in lupus patients. Especially low copy numbers of and deletion of or have consistently been reported as potent risk factors for SLE.2C4 However, the impact of specific genetic backgrounds on certain clinical variants of the disease or its clinical course is scarcely known. In our study, we investigated the contribution of genetic variations of complement to the clinical course of the disease. Since the gene locus is part of the highly polymorphic major histocompatibility complex (MHC) gene region, the impact of other genetic factors located inside the MHC possibly confounding the association of copy number variation and SLE has been discussed controversially. To assess the RAD51 Inhibitor B02 relative weight of genes and potentially confounding genes within the MHC, we analyzed three single-nucleotide polymorphisms (SNPs) recently reported to have the strongest association with SLE in British and Spanish patient groups.3 Material and methods Study participants A total of 169 patients with the clinical diagnosis of SLE based on the 1997 revised American College of Rheumatology (ACR) criteria,5 who presented at the departments of Rheumatology and Nephrology of KRAS the University Hospital of Kiel between 1986 and 2013, were enrolled in this study. Inclusion criteria were a regular and structured follow-up and a treatment period of at least 12 months. The patient group consisted of 160 European individuals, five patients from the Middle East, three patients of Eastern Asian origin and one patient from Togo, Africa. There were 520 age- and sex-matched unrelated controls drawn from the population-based German biobank POPGEN.6 The study was carried out according to the Declaration of Helsinki, written informed consent was obtained from all participants and the study has been approved by the local ethics committee. Ethylenediaminetetraacetate (EDTA) and serum blood samples were taken from all patients and controls. Deoxyribonucleic acid (DNA) preparation and genotyping EDTA blood samples were stored at ?80 up to genetic RAD51 Inhibitor B02 analysis. DNA extraction was performed automatically by the Autopure LS system with Gentra Puregene chemistry (Qiagen, Hilden, Germany). Whole genome amplification was performed using the Illustra GenomiPhi V2 DNA Amplification Kit according to the manufacturers RAD51 Inhibitor B02 guidelines (GE Healthcare, Little Chalfont, UK). copy number genotyping was performed using the common method based on the TaqMan? real-time PCR technology as described elsewhere (assay description: gene locus (assay description: rs2187668: hCV58662585; rs3135391: hCV2455638; rs558702: hCV940258; Life Technologies Corporation, Foster City, CA, USA).3 SNP genotyping was performed using the common TaqMan? SNP Genotyping Assays as described elsewhere.8 concentrations from serum blood samples were measured by nephelometry using the BN II system (Siemens Healthcare, Erlangen, Germany). Quality control Copy number results were assessed using the CopyCaller v1.0 Software (Life Technologies Corporation, Foster City, CA, USA) and were checked for quality according to the recommended procedure obtained from the CopyCaller user manual. In brief, quality control consisted of exclusion of all samples with fewer than three replicates, exclusion of all samples with confidence 0.95 and |CNpredicted C CNcalculated| 0.3, exclusion of all samples with score 2.65 and exclusion of all samples with 2.65 Z score 1.75 and |CNpredicted CCNcalculated| 0.3.9 After quality control, 160 (total for 2 vs. 2 and 5 vs. 5 and for for homozygous deficiency (0 vs. 0), homozygous and heterozygous deficiency (1 vs. 1) and for 3.